Cancer remains a health issue of global importance. Conventional treatment of cancer has involved surgery, radiation and chemotherapy. However, problems with lack of effectiveness and undesirable side-effects remain and more effective therapies are much sought after for those reasons. The detection of tumor-specific molecules to aid in better diagnosis and treatment of cancer is an area of particularly active research. Tumor-specific molecules offer the possibility of targeted therapy using monoclonal antibodies (mAbs) specifically directed against the tumor-specific molecule. However, it has proven difficult to identify tumor-specific molecules.
Growth factors and their receptors play important roles in regulating cell division, proliferation and migration. One prominent family is the epidermal growth factor receptor (EGFR) family, which consists of four members: EGFR, ErbB-2, ErbB-3 and ErbB-4.
The canonical member of the family, EGFR, is the 170 kilodalton (kDa) membrane glycoprotein of the EGFR gene (c-erbB-1). Overexpression of EGFR in human tumors has been intensively studied, and the EGFR gene has been found amplified and over-expressed in a variety of tumors including malignant human gliomas, such as glioblastoma multiforme (GBM). Other members of the EGFR family, such as ErbB-2 and ErbB-3, have also been shown to have a role in cancer. Indeed, the medical importance of this family is highlighted by the success of trastuzumab (Herceptin), a monoclonal antibody that binds to the ErbB-2 receptor, in treating breast cancer.
Seven genomic variants of the EGF receptor (EGFRv) have been identified to date: class I mutants lack the extracellular domain, resembling the v-erbB gene product; class II mutants contain an in-frame deletion of 83 amino acids in the extracellular domain; class III mutants contain an in-frame deletion with a novel junction amino acid; class IV and V mutants contain deletions in the cytoplasmic domain. Class VI and VII mutants contain Class IV and V mutants co-existing with Class III mutants (See Kuan C T., et al., EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr Relat Cancer. 2001 8:83-96.)
Class III mutants (EGFRvIII) are the most frequently detected genomic variant. 40-50% of GBM tumors have EGFR gene amplification. (EGFRvIII was previously known as a Type II sequence; see, for example, U.S. Pat. No. 5,212,290). Of those tumors, 50% express the class III deletion. Class III mutants contain a deletion of exons 2-7 of the gene. Removing exons 2-7 causes an in-frame deletion of 801 base pairs of the coding sequence that removes residues 6 to 273 from the extracellular domain of the wild type EGFR protein. Additionally, removal of exons 2-7 results in an inserted glycine residue at position 6 between the residues 5 and 274, near the N-terminus of the extracellular domain. This deletion creates a unique protein sequence and peptides containing the EGFRvIII-unique sequence have been exploited to generate antibodies specific to the EGFRvIII protein. (Wikstrand C J. et al Investigation of a synthetic peptide as immunogen for a variant epidermal growth factor receptor associated with gliomas. 1993 J. Neuroimmunol 46:165-174; Wikstrand C J, et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res. 1995 55:3140-8; Wikstrand C J, et al. Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res. 1997 57:4130-40; Hills D, et al. Specific targeting of a mutant, activated FGF receptor found in glioblastoma using a monoclonal antibody. Int J. Cancer. 1995 63:537-43).
One peptide, known as pepIII, has been used to immunize mice either alone or conjugated to adjuvants such as keyhole limpet haemocyanin (KLH). PepIII is a 13-mer having the sequence LEEKKGNYVVTDH(SEQ ID NO: 2), which contains the inserted glycine residue (underlined). The peptide may be used alone, or coupled to an adjuvant protein, such keyhole limpet haemocyanin (KLH). A cysteine residue can be attached to pepIII to facilitate conjugation to the adjuvant protein. (See Heimberger A B et al. Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors. Clin Cancer Res. 2003 9: 4247-54.)
Methods for detecting EGFR mutant protein in fluid samples are known in the art; for example, Vogelstein et al. (U.S. Pat. No. 5,212,290) teach the use of a competition based assay. However, direct detection of the protein does not indicate the presence of antibodies directed against EGFRvIII. Detection of mouse antibodies generated against pepIII has been achieved using a standard ELISA assay. (See, for example, U.S. Pat. No. 5,401,828; Heimberger A B et al. supra.) Despite repeated attempts, however, it has not been possible use a standard ELISA assay to detect human anti-EGFRvIII antibodies. Thus, there is a pressing need in the art for an assay that is able to detect human anti-EGFRvIII antibodies reliably.